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1 Figure S1. lev-9 mutants are resistant to levamisole. The levamisole dose-response curves indicate that lev-9 mutants are partially resistant to levamisole similar to lev-10(kr26) mutants. unc-29(x29) mutants, which lack L-AChRs, do not paralyze even at high concentrations. Worms were scored for paralysis after two hours of exposure to levamisole. (mean percentage of paralyzed worm ± SEM, n=4 independent experiments). Figure S2. WAP and CCP domains contain conserved core residues. The WAP domain and the 8 CCP domains of LEV-9 are aligned with consensus sequences obtained from SMART ( conserved cysteine residues are indicated with black boxes. Residues that are conserved in at least 50 % of the domains used to generate the consensus sequences are indicated with grey boxes. (o, alcohol; l, aliphatic;., any residue; a, aromatic; c, charged; h, hydrophobic; p, polar; +, positive; s, small; u, tiny; t, turnlike; -, gap. The grouping of amino acids to classes and class abbreviations used within consensus sequences are described at 1

2 Figure S3. Evolutionary conservation of lev-9. a. Organization of the genomic region containing lev-9. lev-9 is followed by the predicted gene T07H6.4 (Image from Wormbase WS198). Based on in silico translation, this gene would encode a polypeptide containing 8 CCP domains (black circles). b. LEV-9 orthologs are present in ecdysozoans. lev-9 orthologs are readily detected in all sequenced ecdysozoan genomes. They were detected in sequenced genomes using BLAST. In all ecdysozoans, a region homologous to T07H6.4 was located downstream of the predicted lev-9 orthologous sequences. In C. japonica and P. pacificus, the prediction identifies two different genes. In the other species analyzed here, predictions fuse the two ORFs. Whether this actually happens in some species would require experimental confirmation. We separately compared LEV-9 (white box) and T07H6.4 (grey box) aminoacid sequences with the corresponding homologous sequences and it is interesting to note that the amino-acid sequence conservation is systematically higher between LEV-9 and its ortholog than between the putative protein encoded by T07H6.4 and the related one in a given nematode species. In insects, lev-9 orthologs are predicted to contain 1 WAP and 17 CCP domains, but so far, this prediction also lacks experimental support. The conservation of lev-9 and lev-10 in the nematoda phylum, including parasitic species such as Brugia malayi, and in insects strongly suggests that both genes have conserved functions in ecdysozoans. No obvious lev-9 ortholog is detected in deuterostomes. Among vertebrates, only Xenopus tropicalis and fish genomes contain a predicted ORF encoding a WAP-CCP domain protein. However, these predicted proteins only have 2 to 3 CCP domains and amino-acid conservation with LEV- 9 is less than 16 %. Mammalian genomes contain genes encoding multiple CCP domain proteins but not WAP-CCP proteins. However, the WAP domain was not predicted in lev-9 despite the excellent annotation of the C. elegans genome. Therefore, experimental determination of mrna sequences might identify WAP domains in mammalian proteins that are not predicted. It is also possible that the WAP domain was lost during evolution. Assessing phylogenetic relations between CCP-containing proteins and LEV-9 would be hazardous since most of the sequence similarity with LEV-9 arises from conservation of amino-acids involved in the structural fold of CCP domains. Alignments were performed using the EMBOSS needle Pairwise Alignment Algorithm ( Signal peptide sequence was excluded. When only one gene was predicted, we split "Nterm" and "Cterm" regions. Annotation of the P. pacificus genome is still in progress. The LEV-9 sequence was compared to the P.pac California *Assembly Freeze 1* database available at using tblastn. A lev-9 ortholog is present in the contig 58_57. Using Wise2 ( we identified putative exons encoding a putative LEV-9 ortholog. Using the same procedure we identified a putative T07H6.4 ortholog downstream to the lev-9 ortholog. Protein names of nematode species were retrieved from Wormbase (WS198). The protein name of X. tropicalis is retrieved from ensembl database (ENS*: ENSXETP ). 2

3 Figure S4. Mosaic analysis suggests that lev-9 is required in body wall muscle cells. To identify in which cells lev-9 was required for L-AChR function, we performed a mosaic analysis using levamisole sensitivity to measure rescue of the mutant phenotype. A genomic PCR fragment containing lev-9 was coinjected into the germline of lev-9 mutants with two additional plasmids driving GFP expression in neurons or in muscle cells, respectively. In C. elegans, co-injected DNAs form mixed extrachromosomal arrays that are randomly lost at variable frequency during cell division, hence generating somatically mosaic individuals in which some cells carry the transgene (LEV-9(+)) and some do not (LEV-9(-)). Based on GFP expression, we identified rare animals that lost the transgene either in neurons or in muscle. When the transgene was present in most of the muscle cells but absent from neurons, the levamisole response of the mosaic animals was rescued (4 out 4), but not when the transgene was present in neurons and absent from muscles (0 out 23). These data suggested that lev-9 was required in BWM cells for L-AChR function. Figure S5. LEV-9 is a secreted protein. A GFP-LEV-9 fusion protein expressed under the control of the muscle specific promoter Pmyo-3 rescues lev-9(ox177) mutants. It is secreted from BWM cells into the pseudocoelomic cavity and accumulates in coelomocytes (white arrow heads). (Scale bar= 100µm). 3

4 Figure S6. Schematic strategy to insert a T7 tag in the lev-9 locus using the MosTIC technique. Germline expression of the Mos transposase in lev-9(ox177::mos1) animals triggers the excision of the Mos1 transposon present in the first lev-9 exon and causes a DNA double strand break at the excision site (1). The break can then be repaired by homologous recombination using a repair template containing sequence homologous to the broken locus (3,6 kb) provided on an extrachromosomal transgenic array (2). PCR screening identifies animals that inserted the T7 tag sequence present in the repair template into the lev-9 locus (3). Figure S7. Characterization of anti-unc-38 antibodies. Immunostaining of UNC-38 using anti-unc-38 polyclonal antibodies detects L-AChRs in the wild type (a) but not in unc-38(x20) mutants (b) (nr: nerve ring, vc: ventral nerve cord, dc: dorsal nerve cord). Double staining with anti-unc-17 antibodies (c-h) indicates that UNC-38 clusters (c) are juxtaposed to cholinergic release sites (g) in the wild type and are not detected in unc-38(x20) mutants (d). (Scale bar= 10 µm). 4

5 doi: /nature08430 Figure S8. Characterization of anti-acr-16 antibodies. a-b. Immunostaining of ACR-16 using antiacr-16 polyclonal antibodies detects N-AChRs in the wild type (a) but not in acr-16(ok789) null mutants (b) (nr: nerve ring, vc: ventral nerve cord, dc: dorsal nerve cord) (* indicates nonspecific staining of the pharynx in wild type and acr-16 mutants). (Scale bar= 10 µm). c-h. N-AChRs and L-AChRs colocalize at the neuromuscular junction. N-AChRs labeled with anti-acr-16 antibodies (c) and L-AChRs labeled with anti-unc-38 antibodies (e) colocalize in the dorsal cord of a wild-type animal (g). In acr-16(ok789) mutant, no N-AChR is detectable (d) whereas L-AChRs are still clustered (f). (Scale bar= 10 µm). i-n. N-AChRs and GABARs have a different distribution in the nerve cord. N-AChRs labeled with anti-acr-16 antibodies (i) and GABARs labeled with anti-unc-49 antibodies (k) do not colocalize in the dorsal cord of a wild-type animal (m). GABAR distribution is unchanged in acr-16 mutants (l). (Scale bar= 10 µm). 5

6 doi: /nature08430 Figure S9. Generation and characterization of the knock-in strain unc-63::yfp. a. The sequence of the YFP variant Venus was inserted in the unc-63 genomic locus using the MosTIC technique. The Mos1 mutant unc-63(kr19::mos) was used as a starting strain. This allele confers resistance to 1 mm levamisole. A nonrescuing MosTIC template provided the yfp-venus sequence flanked by unc-63 homologous sequences of 3.2 kb and 1.9 kb. Homologous recombination events were identified based on 6

7 recovery of wild-type levamisole sensitivity and subsequently confirmed by genome sequencing. In the unc-63::yfp strain, the YFP Venus is inserted after the residue 383 in the M3-M4 cytoplasmic loop of the UNC-63 L-AChR subunit. (black boxes: unc-63 exons, sp: signal peptide, blue boxes: transmembrane domains) b-c. The AChR subunit UNC-63-YFP is specifically detected by GFP immunostaining in the unc- 63::yfp knock-in strain (b) and not in wild type individuals (c). UNC-63-YFP is present in the nerve ring (nr) and along the dorsal (dc) and ventral nerve cord (vc) of the animals. (Scale bar: 10µm) d. Electrophysiological response of the body wall muscle after pressure ejection of levamisole shows no significant difference between the unc-63::yfp and the wild-type individuals. Black arrows mark the 100ms application onset for 5x10-4 M levamisole. (histograms are average +/- sem, wild type: n=9, unc-63::yfp : n=5) e-m. UNC-63-YFP is clustered at cholinergic neuromuscular junctions. UNC-63-YFP labeled with anti-gfp antibodies in the dorsal nerve cord (e, h) is colocalized with the endogenous L-AChR subunits UNC-29 (f, g) and UNC-38 (i, j) visualized by co-immunostaining. UNC-63-YFP is juxtaposed to the presynaptic cholinergic boutons labeled by immunostaining of the vesicular ACh transporter UNC-17 (l, m) (Scale bar: 10µm). 7